Agents of Bioterrorism

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Agents of Bioterrorism
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Subject Matter Experts at CDC

• Bacillus anthracis - Robbin Weyant
• Brucella spp - Robbin Weyant
• Botulinum toxin - Susan Maslanka
• Francisella tularensis - May Chu
• Hemorrhagic fevers - Tom Ksiazek
• Smallpox - Joe Esposito
• Yersinia pestis - May Chu

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Level A LaboratoryDefinition

• BSL-2 Laboratory with a certified Class II
  biological safety cabinet
• BSL-1 microbiology practices plus
• Directed by competent scientists
• Personnel specifically trained in handling
  pathogenic agents
• Biological safety cabinet, Class II
• Access limited by lab director

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Level A LaboratoryDefinition, cont

• BSL-2 Laboratory with a certified Class II
  biological safety cabinet
• Physical containment practices to minimize
  infectious aerosols
• Sharpsprecautions
• PPE (lab coat, gloves, face shield)
• Biohazard warning signs
• Biosafety manual defining waste/ SH/
  decontamination/surveillance policy/ CC

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Role of the Level A Laboratory

• Rule out critical biological agents
• Refer to higher level laboratory

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Bioterrorism AgentsLaboratory Risk

• Agent BSL Laboratory Risk
• B. anthracis 2 low
• Y. pestis 2 medium
• F. tularensis 2/3 high
• Brucella spp. 2/3 high
• Botulinum toxin 2 medium
• Smallpox 4 high
• Viral Hemorrhagic fever 4 high

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Francisella tularensis

• Tularemia

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Francisella tularensisA Rose by Any Other Name

• Plague-like disease in rodents (California)
• Deer-fly fever (Utah)
• Glandular tick fever (Idaho and Montana)
• Market mens disease (Washington, DC)
• Rabbit fever (Central States)
• OHaras disease (Japan)
• Water-rat trappers disease (Russia)

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Reported Cases of Tularemia - 1990-1998
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Level A ProceduresFrancisella tularensis

• This is a dangerous, highly virulent organism and
  it should not be manipulated at the bench.
  Laboratory-acquired infections can occur easily.
• Gram stain
• Growth characteristics in broth
• Growth characteristics in agar

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Francisella tularensis

• Gram stain
• Poorly staining, tiny gram-negative
• coccobacilli

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Francisella tularensisGrowth Characteristics

• Fastidious, requires cysteine for robust growth
  Cysteine Heart Agar (CHA) is ideal
• Enriched chocolate agar 9 sheep blood
  cysteine
• Not part of Level A routine procedures
• BCYE (for Legionella) also works

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Francisella tularensisGrowth Characteristics

• Will grow initially on sheep and chocolate blood
  agar and
• Thayer-Martin agar, but poorly or not at all
  on passage
• Grows slowly at 35oC, poorly at 28oC

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Francisella tularensisGrowth Characteristicscont
inued

• 24 hours on SBA, CA, TM, CHA
• gray-white, translucent colonies
• usually too small to be seen individually

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Francisella tularensisGrowth Characteristicscont
inued

• 48 hours on SBA, CA, TM, CHA
• SBA - lt1 mm, gray-white, opaque, no hemolysis
• TMA, CA - 1-2 mm,gray-white, flat, entire,
  smooth, shiny
• CHA - 2-4 mm, greenish-white, dense, shiny,
  opalescent sheen

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Gram Negative Coccobacilli

• Most likely
• Acinetobacter (ox.neg)
• Actinobacillus (sticky)
• H. aphrophilus
• Bordetella, Grp. IV (inert, urea pos)
• Pasturella (nonsticky, Mac pos)

• Least likely
• DF-3
• Brucella (Urea pos in seconds - minutes)
• Francisella (Urea neg)

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Francisella tularensisRapid Method Results

• Not on the data base of MicroScan or Vitek or API
• Should not be worked with in the Level A lab

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Tularemia

• Contagious --- no
• Infective dose --- 10-50 organisms
• Incubation period --- 1-21 days (average3-5
  days)
• Duration of illness --- 2 weeks
• Mortality --- treated low untreated
  moderate
• Persistence of organism ---months in moist soil
• Vaccine efficacy --- good, 80

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Francisella tularensisTechnical Hints
If you see

• Tiny, gram-negative coccobacilli from blood,
  lymph node aspirate, or respiratory specimens
• Blood isolates that will grow slowly on chocolate
  agar but poorly or not at all on blood agar in 24
  hours
• Faint growth in thio requires cysteine in other
  broth

Refer
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Yersinia pestis

• Plague

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Plague Epidemiology

• U.S. averages 13 cases/yr (10 in 1998)
• 30 of cases are in Native Americans in the
  Southwest. 15 case fatality rate

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Plague Epidemiologycontinued

• Most cases occur in summer and near the patients
  residence
• bubonic (infected lymph nodes)
• septicemic (blood-borne organisms)
• pneumonic (transmissible by aerosol deadliest)

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Yersinia pestisSpecimen Selection

• Specimen selection is important
• Bubonic - bubo - lymph node aspirate
• Septicemic - blood - organisms may be
  intermittent. Take three specimens 10-30 minutes
  apart
• Pneumonic
• Sputum/throat - use Wayson and DFA stain
• Bronchial washings - Wayson and DFA stain

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Yersinia pestisspecimen inoculation

• Inoculate routine plating media and make thin
  smear for DFA
• Use Wayson only if DFA is unavailable

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Level A ProceduresYersinia pestis

• Gram stain
• Wayson stain
• Growth characteristics on agar
• Growth characteristics in broth

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Yersinia pestisGram stain

• Small, gram-negative bipolar-stained coccobacilli

Must confirm by DFA and mouse inoculation
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Yersinia pestisWayson Stain

• Used for rapid assessment
• for specimens when DFA is not available
• when it is a part of the identification process
• Best with tissue, sputum, blood
• Stains of pure culture isolates tend to lose
  bipolarity

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Yersinia pestisWayson Stain

• Pink-blue cells with a closed safety pin look

Wayson stain alone is not diagnostic
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Yersinia pestisGrowth in Broth

• Brain Heart Infusion Broth (two tubes)
• Incubate at 28oC (best) and 37oC for
• 24-48h
• Do not shake tubes
• Observe suspended flocculent clumps like
  stalactites on side and bottom of tube. Broth
  remains clear

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Yersinia pestis in Broth

Y. pestis
Y. pseudotuberculosis
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Yersinia pestis Growth on Agar

• Sheep blood agar - 28oC (faster)
• and 37oC (for DFA tests)
• Looks like other enterics

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Yersinia pestisRapid Method Results

• On the data base of MicroScan, Vitek, and API 20E
• True accuracy not yet determined

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Yersinia pestisTechnical Hints

• Small gram-negative, poorly staining rods from
  blood, lymph node aspirate, or respiratory
  specimens
• Safety pin appearance in Gram, Wright, Giemsa, or
  Wayson stain
• More than one patient in a short, specified
  period with fever, lymphadenopathy

Refer
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Variola virus

• Smallpox

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VariolaSmallpox virus

• Large DNA virus

• Dumbbell-shaped core

• Complex membranes

Refer
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VariolaSmallpox virus

• The family Poxviridae consists of eight genera
  and a few unclassified species
• Two species are human viruses
• Variola virus (genus Orthopoxvirus)
• Molluscum contagiosum virus (genus
  Molluscipoxvirus)
• Orthopoxvirus includes vaccinia (a lab virus),
  monkeypox, cowpox, and buffalopox

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Smallpox virus

• Stored stocks to be retained until 2002 by U.S.
  and Russia
• Undeclared virus could be anywhere
• No cases in over 20 years
• Controversial decision
• Immunity lost in U.S. population
• Highly vulnerable to infection

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Level A ProceduresSmallpox virus

• Rule out chickenpox (PCR)!
• Specimen of choice is lesion material from
  pustules.
• Collect vesicular fluid from each single lesion
• Place droplet fluid as a drop on a clean slide -
  Do not smear
• Store each slide in separate slide holder
• Capillary tubes or dry swabs are alternatives

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Level A ProceduresSmallpox virus

• Autopsy specimens must be frozen.
  Formalin fixation is OK for histopathologic study
• Contact CDC for approval to ship
• Send slide in a non-breakable holder. Do not use
  a transport fluid.
• Store at 4oC briefly or at -20oC to -70oC
• Decontaminate with 0.5 hypochlorite

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Hemorrhagic Fever Viruses
Marburg
Ebola
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Hemorrhagic Fever Viruses

• Arenaviruses
• Argentine HF
• Bolivian HF
• Sabia Associated HF
• Lassa fever
• Lymphocytic choriomeningitis
• Venezuelan HF

• Bunyaviruses
• Crimean-Congo HF
• Rift Valley Fever
• Hantavirus Pulmonary Syndrome HF with 0Renal
  Syndrome

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Hemorrhagic Fever Viruses (continued)

• Filoviruses
• Ebola HF
• Marburg HF

• Flaviviruses
• Tick-borne Encephalitis
• Kyasanur Forest Disease Omsk HF

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Viral Hemorrhagic Fevers

• Contagious --- Moderate
• Infective dose --- 1-10 particles
• Incubation period --- 4-21 days
• Duration of illness --- 7-16 days
• Mortality ---variable
• Persistence of organism --- unstable
• Non-endemic in U.S.
• Vaccine efficacy --- no vaccine

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VHF Specimens

• Diagnosis is clinical, not laboratory

Refer
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Handling VHF Specimens

• No specimen accepted without prior consultation -
  404-639-1115
• Serology - 10-12 ml 5 ml minimum
• serum drawn at admission (acute, convalescent 21
  days later post-mortem heart blood
• ship serum cold or on dry ice in a plastic tube

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Handling VHF Specimens-Immunohistochemistry

• Prefer lung, kidney, spleen tissue
• Other lymph node, heart, pancreas,
• pituitary, brain, liver
• Paraffin blocks preferred. Formalin-fixed tissue
  acceptable
• Ship blocks/tissue at RT - do not freeze.
  Autopsy/surgical report required.

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Handling VHF Specimens-PCR/Virus isolation

• Ante-mortem - biopsy of lung or bone marrow
  aspirate or clot
• Post-mortem - spleen, lung, kidney, liver, nodes,
  heart, pancreas, pituitary, brain, liver
• Must be at least 1 cm3
• Buffy coat, clot, tissue - dry ice

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Brucella spp.

• Brucellosis

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BRUCELLOSIS

• A zoonotic disease caused by any of 4 Brucella
  sp. abortus, melitensis, suis, and canis
• A systemic infection characterized by an undulant
  fever pattern
• But relatively rare in the U.S. with
  approximately 100 cases/yr

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BRUCELLOSIS HISTORY

• 1887 Bruce - Malta fever, M. melitensis
• 1897 Bang - cattle abortion, B. abortus
• 1914 Traum - sow, B. suis
• 1920 Evans, Meyer, Shaw - Brucella
• 1954 B. suis, first weaponized U.S. agent
• 1968 Carmichael - Beagles, B. canis

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BRUCELLOSISTRANSMISSION

• Unpasteurized dairy products
• The most common mode of transmission
• Direct skin contact
• Occupational hazard for farmers, butchers,
  veterinarians, and laboratory personnel
• Aerosols
• Highly infectious

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BRUCELLOSIS

• Infective dose 10 -100 organisms
• Incubation period 5 days - gt 6 months
• Duration of illness weeks to months
• Fever, profuse sweating, malaise, headache and
  muscle/back pain.
• Person to person transmission no
• Mortality lt5
• Persistence of organism very stable